Antibodies are specific immunoglobulin molecules produced by vertebrate immune systems in response to challenge by foreign proteins, glycoproteins, cells, or other typically foreign substances. The sequence of events which permits an organism to overcome invasion by foreign cells or to rid the system of foreign substances is at least partially understood. An important part of this process is the manufacture of antibodies which bind specifically to a particular foreign antigenic substance. The binding specificity of such polypeptides to a particular antigen is highly refined, and the multitude of specificities capable of being generated by an individual vertebrate is remarkable in its complexity and variability. Thousands of antigens are capable of eliciting antibody responses, each almost exclusively directed to the particular antigen which elicited it.
Immunoglobulins include both antibodies, as above described, and analogous protein substances which lack antigen specificity. The latter are produced at low levels by the lymph system and in increased levels by myelomas.
Antibodies are produced by B lymphocytes and represent the humoral arm of the immune defense system. Because of their antigen specificity, antibodies comprise numerous diagnostic and therapeutic applications. For example, they can be used as specific immunoprecipitating agents to detect the presence of an antigen which they specifically bind by coupling the antigen-antibody reaction with suitable detection techniques such as labeling with radioisotopes or with detectable enzymes (RIA, EMIT, and ELISA). Antibodies are thus the foundation of immunodiagnostic tests for many antigenic substances.
Another important application of antibodies involves their use as therapeutics. The therapeutic administration of antibodies has recently been described for the treatment of numerous disease conditions including cancer, and numerous infectious diseases.
The therapeutic usage of antibodies has been the focus of greater interest since the development of monoclonal antibody/hybridoma technology by Kohler and Milstein (Proc. Natl. Acad. Sci., USA, 77:2197 (1980)). Monoclonal antibodies, which are produced by hybridomas, are preferable to polyclonal antibodies because of their greater antigenic specificity. Monoclonal antibodies have a lesser tendency than polyclonal antibodies to non-specifically bind to non-targeted moieties, e.g., cells which do not express the corresponding antigen. However, monoclonal antibodies still suffer from some disadvantages, e.g., they tend to be contaminated with other proteins and cellular materials of hybridoma (mammalian) origin. Also, hybridoma cell lines tend to be unstable and may alter the production of the antibody produced or stop secreting the antibody altogether.
In an effort to obviate some of the problems associated with polyclonal and monoclonal antibodies, and further to obtain a reproducible supply of antibodies having a defined binding specificity, researchers have used recombinant techniques to produce immunoglobulins which are analogous or modified in comparison to antibodies normally found in vertebrate systems. For example, U.S. Pat. No. 4,816,397 issued on Mar. 28, 1989 to Boss et al. and U.S. Pat. No. 4,816,567 issued on Mar. 28, 1989 to Cabilly et al. disclose recombinant immunoglobulins and immunoglobulin fragments, and methods for their production.
To enhance or modify the properties of recombinant antibodies, it is further known to produce mutant or chimeric antibodies, e.g., which comprise sequences from several different mammalian species or bispecific antibodies which comprise antigenic binding sequences from two different antibodies. For example, humanized antibodies which comprise antigen-binding sites from a non-human species (typically murine) but wherein the remainder of the immunoglobulin is of human origin are known in the art, and have been reported to have significant potential as therapeutics because of their reduced antigenicity. It is further known to produce recombinant antibodies of single chain form, which completely lack constant domain sequences but which bind antigen. (See, Bird et al., Science, 242, 423-426 (1988)).
In order to increase the efficacy of antibody molecules as diagnostic or therapeutic agents, it is conventional to covalently bind or complex desired molecules thereto, in particular effector or reporter molecules. Effector molecules essentially comprise molecules having a desired activity, e.g., cytotoxic activity. By contrast, a reporter molecule is defined as any moiety which may be detected using an assay. Examples of effector molecules which have been attached to antibodies include by way of example, toxins, anti-tumor agents, therapeutic enzymes, radionuclides, antiviral agents, chelating agents, cytokines, growth factors, and polynucleotides. Examples of reporter molecules which have been conjugated to antibodies include, by way of example, enzymes, radiolabels, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, and colored particles.
While it is desirable to attach molecules to antibodies in order to impart a desired activity to the antibody or provide for the detection thereof, the attachment of desired molecules to antibodies is not always possible to carry out conveniently, or effectively, because such attachment may result in loss of antibody activity. In particular, current methods for generating radiolabeled antibodies for diagnostic and therapeutic use suffer from such limitations. For example, the ratio of target-specific versus non-specific uptake of radiolabeled antibodies used in tumor imaging is often low, resulting in unclear images or missing tumor sites. Moreover, the low therapeutic index of radiolabeled antibodies limits the use of high radiation doses in radiation therapy.
The underlying reason for such problems is largely because the labeling chemistry for introduction of the radiolabel results in the partial denaturation of the antibody structure, which in turn causes the antibodies to aggregate in vivo or in vitro. Aggregated and damaged immunoglobulins are recognized by scavenger cells in the body, such as macrophages and Kupffer cells in the liver and lung.
Another problem is that most coupling strategies result in non site-specific attachment of the molecule to the antibody molecule, in particular, attachment may occur at antibody residues which are essential for antigen binding or other antibody functions. For instance, a known site of attachment of desired molecules to antibody molecules comprise thiol groups, since thiol groups occur naturally in proteins as cysteine residues. However, such residues are relatively uncommon, are often inside the molecule and are frequently involved in forming disulfide bridges within or between protein molecules. Thus, there is a danger that if a naturally occurring cysteine residue is used as a site of attachment, it will interfere with the normal folding and stabilization of the antibody protein.
In an effort to obviate such problems, alternative strategies have been developed which provide for site-selective attachment of a desired molecules to antibodies, without loss of antigen-binding activity. For example, it is known to produce recombinant antibodies comprising cysteine residues introduced into their surface structure to provide a thiol group which is available for covalent binding to an effector or reporter molecule. This method has been reported to facilitate the site-specific attachment of desired molecules without loss of antigen binding properties. (See, U.S. Pat. No. 5,219,996 issued on Jun. 15, 1993 to Bodmer et al.) However, this is not always possible or convenient since it obviously requires the possession of a recombinant DNA encoding the particular antibody.
It has further been proposed to derivatize immunoglobulins by selectively introducing sulfhydryl groups in the Fc region of an immunoglobulin, using reaction conditions which purportedly do not result in alteration of the antibody combining site. Antibody conjugates produced according to this methodology are disclosed to exhibit improved longevity, specificity and sensitivity (U.S. Pat. No. 5,196,066 issued on Mar. 2, 1993 to Bieniarz et al.).
Site-specific attachment of effector or reporter molecules, wherein the reporter or effector molecule is conjugated to a carbohydrate residue in the Fc region has also been disclosed in the literature. (See, e.g., O'Shannessy et al., J. Immun. Meth., 99, 153-161 (1987)). This approach has been reported to produce diagnostically and therapeutically promising antibodies which are currently in clinical evaluation.
Another known method of site-specific attachment of molecules to antibodies comprises the reaction of antibodies with hapten-based affinity labels. Essentially, hapten-based affinity labels react with amino acids in the antigen binding site, thereby destroying this site and blocking specific antigen reaction. However, this is disadvantageous since it results in loss of antigen binding by the antibody conjugate.
Thus, based on the foregoing, it is clear that there still exists a significant need in the art for improved methods of attaching molecules to antibodies, in particular effector or reporter molecules, which are site-specific and which moreover result in antibody conjugates having substantially unaltered structure and biological activity, most especially antigen binding activity.
Molecules containing azido groups have been shown to form covalent bonds to proteins through reactive nitrene intermediates, generated by low intensity ultraviolet light. Potter & Haley, Meth. in Enzymol., 91, 613-633 (1983). In particular, 2- and 8- azido analogues of purine nucleotides have been used as site directed photoprobes to identify nucleotide binding proteins in crude cell extracts. Owens & Haley, J. Biol. Chem., 259:14843-14848 (1987); Atherton et al., Biol. of Reproduction, 32, 155-171 (1985). The 2- and 8-azido nucleotides have also been used to map nucleotide binding domains of purified proteins. Khatoon et al., Ann. of Neurology, 26, 210-219 (1989); King et al., J. Biol. Chem., 269, 10210-10218 (1989); and Dholakia et al., J. Biol. Chem., 264, 20638-20642 (1989).
Photoaffinity probes have been used to determine specific nucleotide binding sites on a biologically active recombinant peptide molecule. Campbell et al., PNAS, 87, 1243-1246 (1990). The probes have also been used to study enzyme kinetics of purified proteins. Kim et al., J. Biol. Chem., 265, 3636-3641 (1990).
Recently, ATP or GTP analog photoaffinity labeled probes have been used to detect a glutamine synthetase nucleotide binding protein having an apparent molecular weight of about 42,000 proteins to aid in the diagnosis of Alzheimer's disease in a mammal. U.S. Ser. No. 08/138,109 now U.S. Pat. No. 5,445,937 filed on Oct. 20, 1993 by Haley et al. Additionally, ATP or GTP analog photoaffinity-labeling reagents have been disclosed for use in the detection of particular nucleotide binding proteins to aid in the diagnosis of cancer in a mammal and in the diagnosis of leukemia in a mammal. (Id.)
However, while it had been previously known to use nucleotide photoaffinity probes, and specifically purine containing photoaffinity analogs (GTP- and ATP-analogs), to map nucleotide binding domains of purified proteins and to identify specific nucleotide binding sites on recombinant peptide molecules, the use of nucleotide photoaffinity probes to label antibodies has not been previously reported in the literature. This is essentially because it had not been previously known that antibody molecules comprise nucleotide photoaffinity sites, and in particular, sites having high affinity for purine, azidopurine and other similar heterocyclic bases, which may be efficiently photolabeled using appropriate photoaffinity probes.